The spatial arrangement of thalamic nuclei is important for generating the precise topographical relationship needed to fulfill its role as a relay and information processing center. Despite advances in our understanding of the early events regulating thalamic growth and regionalization, there remain major gaps in knowledge of the mechanisms by which heterogeneous clusters of excitatory relay neurons are specified and aggregate into distinct thalamic nuclei. One particular challenge has been to decipher the full complement of thalamic progenitor identities and to elucidate their contribution to specific thalamic nuclei. The overarching goal of this grant proposal is to elucidate the molecular logic underlying the identity of neuronal progenitor sub-types in the developing thalamus and to gain mechanistic insight into how alterations in their gene regulatory networks contribute to sensory and motor deficits in neurodevelopmental disorders. One factor in particular, the secreted morphogen Sonic hedgehog (Shh), has been implicated in spatiotemporal and threshold models of thalamic development that differ from other areas of the CNS due, in large part, to its expression within two signaling centers, the basal plate and the zona limitans intrathalamica (ZLI), a dorsally projecting spike that separates the thalamus from the prethalamic territory. We will employ a top down, phenotype driven, approach to deconstruct the spatiotemporal role of Sonic hedgehog (Shh) signaling in thalamic development and function. Mice with mutations in Shh brain enhancers (SBE1 and SBE5) will be leveraged to investigate the effect that disruption of Shh expression has on the formation of thalamic nuclei using a multiplexed single molecule in situ method for quantitative analysis of gene expression in individual thalamic cell types. Follow up studies will examine the impact that these neuroanatomical defects have on thalamic circuit assembly, as well as motor and sensory behaviors. A parallel, bottom-up approach, will define the genetic programs driving thalamic progenitor subtype identity and corresponding lineage trajectories using single-cell RNA-seq (scRNA-seq) profiling of control and SBE1/5-/- mutant embryos over developmental time. Finally, we will also study the mechanism of enhancer redundancy and the role that SBE1 and SBE5 play in facilitating the three-dimensional folding dynamics of the Shh locus in the developing brain using a novel super resolution imaging-based method. Taken together, the conceptual framework of our experimental approach is intended to address fundamental questions of biological importance in developmental neurobiology using innovative methods with the added goal of uncovering novel pathogenic mechanisms of neurodevelopmental disease that may inform future treatment options.